Fig. 2: Targeted coordinate patterns (TCP) in sequential iterations for imaging and fast single-molecule tracking.

a The TCP defines a set of relative coordinates that are used to probe the position of the fluorescent molecule (star) in the sample. In a typical 2D MINFLUX iteration, the central zero of the donut-shaped excitation beam is targeted to these coordinates that are arranged in a hexagon with diameter L plus an optional midpoint (all in green). After each iteration, the TCP is re-centered based on the prior estimate of the molecular position. A MINFLUX sequence defines the parameters of these consecutive localization iterations, starting with a pre-scan, followed by intermediate and final iterations which are processed during individual localization events. Scale bar: 150 nm. b–d Typical parameters and estimator performances for imaging and tracking applications. While imaging sequences tend to spend more photons on later iterations to maximize precision, tracking sequences are optimized for speed and thus spend only minimal photons on the final iteration, which tracks the molecule (d). Simulated molecules at positions (crosses) within the FOV and the distributions (red) of their respective estimate represented by their mean (dot) and confidence intervals (ellipsoids). Note the nonvanishing residual bias at the outmost grid locations which marks the edge of the usable field of view. In the tracking parts of (c, d), every second confidence interval is shown for clarity of display. Source data are provided as a Source Data file. Scale bars: 100 nm (b), 50 nm (c, d).